Live vaccines and methods of treatment therewith

ABSTRACT

Disclosed herein are methods and pharmaceutical formulations for administering vaccines to birds. In preferred embodiments, the invention provides methods of administering live pathogenic virus vaccines to birds in ovo, more preferably, during the last quarter of in ovo incubation. Interferon, more preferably, Type I interferon, may be advantageously administered in conjunction with live virus vaccines to decrease the pathogenicity thereof. Interferon must be provided at doses sufficient to protect against pathogenicity of the live vaccine, but not at doses so high as to prevent the host from mounting an active immune response. Further provided are pharmaceutical formulations comprising effective doses of live vaccine and interferon. Finally, the present invention provides methods of administering interferon together with live vaccines to young avians to effectively overcome the interfering effects of maternal antibodies.

RELATED APPLICATION INFORMATION

This application claims the benefit of U.S. Provisional Application No.60/082,196 filed Apr. 17, 1998, which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods for protecting avians againstdisease, in particular methods of administering vaccines to avians.

BACKGROUND OF THE INVENTION

Newcastle disease (ND) causes global economic losses for the poultryindustry in the range of 40 million dollars annually. The disease iscaused by several different RNA viruses from the Paramyxoviridae familyand symptoms range from subclinical disease to high mortality. Althoughvaccination programs can control ND, there are still problems due toadverse vaccine reactions and requirements for multiple vaccineadministrations.

Chicks raised in the commercial poultry industry typically arevaccinated against multiple diseases. In the past, immunization for NDVgenerally occurred at day one and day fourteen post-hatch. Morerecently, in ovo injection devices have automated immunization, allowingtreatment of the embryos prior to hatch. However, thus far, there hasbeen little success with in ovo administration of live viral vaccineswithout a high incidence of embryo mortality. Use of a virulent NDV orother viral vaccine strain capable of producing a protective immuneresponse with one in ovo administration would be highly advantageous.However, in ovo NDV live virus vaccination is usually toxic to theembryo, and birds that do hatch from in ovo vaccinated eggs exhibit highearly mortality. Ahmad & Sharma, (1993) Avian Diseases 37:485.

Accordingly, there remains a need in the art for safe and efficaciousmethods of administering live pathogenic virus vaccines to birds in ovo.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that interferon can beadministered in conjunction with vaccines to decrease the pathogenicitythereof. In particular, interferons are effective in decreasing thepathogenic effects of live vaccines in embryonic birds. Accordingly, thepresent invention provides methods and pharmaceutical formulations foradministering live pathogenic vaccines, preferably live pathogenic virusvaccines, to birds in ovo. The dose of interferon must be sufficient toprotect the subject from the pathogenic effects of the live vaccines,but should not be so high as to prevent infection by the vaccine.

In addition, the present investigations have led to the discovery thatadministration of interferon in conjunction with vaccines to birds inovo and hatchlings can overcome the inactivating (i. e., neutralizing)effects of maternal antibodies. It is well-known in the art thatmaternally-transmitted antibodies interfere with the efficacy of earlyvaccination programs in young birds. Accordingly, the present inventionprovides methods and pharmaceutical formulations for effectivelyvaccinating avian embryos and young maternal antibody positive avians.

In one embodiment, the present invention provides a method of producingprotective immunity against a viral disease in an avian subject,comprising: (a) administering to an avian subject in ovo a compositioncomprising a vaccine comprising a live pathogenic virus; and (b)administering to the avian subject in ovo a composition comprisinginterferon; wherein the live pathogenic virus is administered in anamount effective to produce an immune response in the avian subject; andwherein the interferon is administered in an amount effective to (1)protect the avian subject from pathology that would occur in the absenceof the interferon due to the administration of the vaccine, and (2)allow the production of a protective immune response in the aviansubject.

As a further aspect, the present invention provides a method ofproducing protective immunity against Newcastle disease in a chicken,comprising: (a) administering to a chicken during the last half of inovo incubation a composition comprising a vaccine comprising a livepathogenic Newcastle disease virus; and (b) administering to a chickenduring the last half of in ovo incubation a composition comprising aType I interferon; wherein the live pathogenic virus is administered inan amount effective to produce an immune response in the chicken; andwherein the Type I interferon is administered in an amount effective to(1) protect the chicken from pathology that would occur in the absenceof the Type I interferon due to the administration of the vaccine, and(2) allow the production of a protective immune response in the chicken.

As a further embodiment, the present invention provides a method ofreducing mortality from the administration of a live vaccine virus inovo to an avian subject, comprising: (a) administering to an aviansubject in ovo a composition comprising a vaccine comprising a livevaccine virus; and (b) administering to the avian subject in ovo acomposition comprising interferon; wherein the live vaccine virus isadministered in an amount effective to produce an immune response in theavian subject; and wherein the interferon is administered in an amounteffective to (1) protect the avian subject from pathology that wouldoccur in the absence of the interferon due to the administration of thevaccine, and (2) allow the production of a protective immune response inthe avian subject.

As still a further aspect, the present invention provides a method ofreducing disease from the administration of a live vaccine virus in ovoto an avian subject, comprising: (a) administering to an avian subjectin ovo a composition comprising a vaccine comprising a live vaccinevirus; and (b) administering to the avian subject in ovo a compositioncomprising interferon; wherein the live vaccine virus is administered inan amount effective to produce an immune response in the avian subject;and wherein the interferon is administered in an amount effective to (1)protect the avian subject from pathology that would occur in the absenceof the interferon due to the administration of the vaccine, and (2)allow the production of a protective immune response in the aviansubject.

As yet a further aspect, the present invention provides a method ofproducing protective immunity against a viral disease in an aviansubject, the method comprising administering to an avian subject duringthe last quarter of in ovo incubation a composition comprising a vaccinecomprising a live pathogenic virus and interferon, wherein the livepathogenic virus is administered in an amount effective to produce animmune response in the avian subject; and wherein the interferon isadministered in an amount effective to (1) protect the avian subjectfrom pathology that would occur in the absence of the interferon due tothe administration of the vaccine, and (2) allow the production of aprotective immune response in the avian subject.

Pharmaceutical formulations comprising a composition comprising avaccine comprising a live pathogenic virus and interferon in apharmaceutically-acceptable carrier are also an aspect of the invention.

As yet a further aspect, the present invention provides a method ofproducing protective immunity against a viral disease in an aviansubject, comprising: (a) administering to an avian subject during thefirst month post-hatch a composition comprising a vaccine comprising alive pathogenic virus; and (b) administering to the avian subject duringthe first month post-hatch a composition comprising interferon; whereinthe live pathogenic virus is administered in an amount effective toproduce an immune response in the avian subject; and wherein theinterferon is administered in an amount effective to (1) protect theavian subject from pathology that would occur in the absence of theinterferon due to the administration of the vaccine, and (2) allow theproduction of a protective immune response in the avian subject.

These and other aspects of the present invention will be set forth inmore detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the effects of in ovo NDVvaccine dose on hatchability of SPF chicken embryos. Embryonic day 18eggs were administered either PBS or a 10⁴, 10², 1 or 10⁻² EID₅₀ dose ofNDV vaccine, and hatchability of each treatment group was monitored.There were 40 eggs per treatment group.

FIG. 2 is a graphical representation of the effects of in ovo NDV doseon 7-day post-hatch mortality of SPF chicken embryos. These data werecollected as part of the same study presented in FIG. 1. Eggs wereadministered either PBS or a 10⁴, 10², 1 or 10⁻² EID₅₀ dose of NDVvaccine on embryonic day 18, and survivability was monitored for 7 dayspost-hatch. There were 40 eggs per treatment group.

FIG. 3 is a graphical representation of the effects of IFN-Iadministration in conjunction with NDV vaccination in ovo onhatchability of SPF chicken eggs. On embryonic day 18, eggs wereco-administered a 1 EID₅₀ dose of NDV vaccine together with PBS or 0.25,2.5 or 25 μg IFN-I, and hatchability was monitored for each treatmentgroup. There were 60 eggs per treatment. Controls received PBS alone.

FIG. 4 is a graphical representation of the effects of IFN-Iadministration in conjunction with NDV vaccination in ovo on 8-daypost-hatch survival of SPF chicks. These data were collected as part ofthe same study presented in FIG. 3. On embryonic day 18, eggs wereco-administered a 1 EID₅₀ dose of NDV vaccine together with PBS or 0.25,2.5 or 25 μg IFN-I, and survivability was monitored for 8 days afterhatch. There were 60 eggs per treatment. Controls received PBS alone.Data are inclusive of embryonic mortality.

FIG. 5 is a graphical representation of the effects of IFN-Iadministration in conjunction with NDV vaccination in ovo onhatchability of SPF chicken eggs. Data were collected from threeseparate trials with 25 to 40 eggs per treatment group, depending on thetrial. All treatments received NDV vaccine at a 10 EID₅₀ dose and eitherno IFN-I (treatment 1) or 0.2, 2.0 or 20 μg IFN-I (treatments 2-4,respectively) in ovo. Hatchability was monitored for each treatmentgroup. The results for each treatment were averaged across the threetrials.

FIG. 6 is a graphical representation of the effects of IFN-Iadministration in conjunction with NDV vaccination in ovo onhatchability of SPF chicken eggs. Day 18 embryonic eggs wereadministered 10 EID₅₀ NDV vaccine with PBS or 0.1, 0.2, 1.0, 2.0, 10 or20 μg IFN-I per egg. There were 32 eggs per treatment group.

FIG. 7 is a graphical representation of the effects of IFN-Iadministration in conjunction with NDV vaccination in ovo onhatchability of SPF chicken embryos. Day 18 embryonic eggs wereadministered 10 EID₅₀ NDV vaccine together with 0, 20 or 40 μg IFN-I peregg. Two different IFN-I preparations were assessed in this study. Onetreatment group received PBS alone (positive control) There were 27 eggsper treatment group.

FIG. 8 is a graphical representation of the effects of IFN-Iadministration in conjunction with increasing doses of NDV vaccine inovo on hatchability of SPF chicken embryos. Embryonic day 18 eggs wereadministered 15 μg IFN-I together with 10, 10², or 10³ EID₅₀ NDV, andhatchability was monitored for each treatment group. The positivecontrol group received PBS alone. There were 47 eggs per treatmentgroup. In addition, a comparison was performed between administration ofHPLC purified IFN-I versus non-HPLC purified IFN-I in the presence of 10EID₅₀ NDV.

FIG. 9 is a graphical representation of the effects of IFN-Iadministration with increasing doses of NDV vaccine in ovo onhatchability of SPF chicken embryos. Embryonic day 18 eggs wereco-administered 20, 30 or 50 μg IFN-I per egg in conjunction with 10,10² or 10³ EID₅₀ NDV, and hatchability was monitored for each treatmentgroup. There were 40 eggs per treatment group.

FIG. 10 is a graphical representation of the effects of IFN-Ico-administration with NDV vaccination in ovo on 7-day post-hatchsurvivability of SPF chicken embryos. Embryonic day 18 eggs wereco-administered 0, 5, 15, 30 or 45 μg IFN-I per egg together with 10EID₅₀ NDV vaccine, and survivability for each treatment group wasmonitored for 7 days following hatch. One treatment group only receivedPBS (positive control). There were 60 eggs per treatment group.

FIG. 11 is a graphical representation of the effects of IFN-Ico-administration with NDV vaccination in ovo on 7-day post-hatchsurvivability of SPF chicken embryos. Embryonic day 18 eggs wereco-administered 0, 5, or 20 μg IFN-I per egg together with 10, 10² or10³ EID₅₀ NDV vaccine, and survivability was monitored for 7 daysfollowing hatch. One treatment group only received PBS (positivecontrol). There were 43 eggs per treatment group.

FIG. 12 is a graphical representation of the data from FIG. 11 showingonly the treatment groups receiving 20 μg IFN-I per egg together with10, 10² or 10³ EID₅₀ NDV vaccine, as well as the positive control (PBS)group.

FIG. 13 is a graphical representation of the effects ofco-administration of IFN-I and NDV vaccine in ovo on hatchability and14-day survivability of commercial broilers. The positive control onlyreceived PBS. Embryonic day 18 eggs were co-administered with 0, 20 or40 μg IFN-I per egg in conjunction with 0, 10², 10³ or 10⁴ EID₅₀ NDVvaccine. Hatchability and 14-day post-hatch survivability were monitoredfor each treatment group. There were 60 eggs per treatment group.

FIG. 14 is a graphical representation of the effects ofco-administration of IFN-I and NDV vaccine in ovo on hatchability ofcommercial broilers. The positive control only received PBS. Embryonicday 18 eggs were co-administered with 0, 10, 20 or 30 μg IFN-I per eggin conjunction with 0, 10^(2.5) or 10^(3.5) EID₅₀ NDV vaccine.Hatchability was monitored for each treatment group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and pharmaceutical formulationsfor administering live virus vaccines to birds in ovo. The invention isbased, in part, upon the discovery that administration of interferon(IFN), in particular Type I interferon (IFN-I), can protect birds fromthe pathology and mortality associated with administration of live virusvaccines to bird embryos. Prior to the present investigations, vaccinesagainst Marek's Disease and bursal Disease were the only live viralvaccines that could be administered in ovo without a high incidence ofembryo mortality. The invention is further based on the discovery thatadministration of IFN, in particular IFN-I, in conjunction withvaccination with live virus vaccines pre- or post-hatch provides a meansto effectively vaccinate birds in the presence of interfering maternalantibodies. Furthermore, the present invention provides pharmaceuticalformulations and methods for administering live virus vaccines (i.e., toproduce active immunity against the virus) in conjunction with IFN tobirds in ovo, without causing substantial disease or death (either pre-or post-hatch) among the vaccinated birds.

A. Interferon

Interferon for use in the present invention can be IFN-I and/or IFN-II,with IFN-I being preferred. IFN-I is a family of closely-relatedproteins that are produced by leucocytes (α subtypes), fibroblasts (βsubtypes), lymphocytes (IFNω), and ruminant embryos (IFNτ). Robert J.Donnelly, The Type I (α/β/ω/τ) Interferon Family, in Guidebook toCytokines and Their Receptors 111 (Nicos A. Nicola ed., 1994). The term“interferon” as used herein encompasses biologically-active IFN analogsand derivatives (e.g., can protect an avian subject from the pathogeniceffects of a live vaccine, as described herein, or alternatively,possesses any other known biological action of IFN), as well asbiologically-active truncated IFN molecules, as are known by those ofskill in the art. The IFN can be recombinant or purified from naturalsources, with recombinant being preferred. Additionally, the IFN can bepurified by any method known in the art. Finally, the IFN can be fromany species of origin, including avian and mammalian IFNs, for example,chicken, turkey, murine, human, and bovine IFN. Avian IFNs are preferredfor administration to avian subjects, with chicken and turkey IFN beingmore preferred, and chicken IFN being most preferred. Mammalian IFNs arepreferred for administration to mammalian subjects, with human, bovine,and murine IFNs being more preferred. In general, it is preferred toadminister IFN derived from the same species as the subject.

According to the present invention, IFN is incorporated inpharmaceutical formulations and administered in an amount effective toreduce (i.e., ameliorate, delay, diminish, and/or decrease) thepathogenic effects (e.g., disease, mortality, etc.) caused to the avianembryo by the in ovo administration of the live pathogenic virusvaccine, without blocking the production of a protective immune responsein the bird. By “reduce”, it is not meant that there be no detrimentaleffects from the virus vaccine. The IFN ameliorates the pathogeniceffects of the virus vaccine, such that the benefits of vaccinationoutweigh the detriments. Alternatively stated, the IFN willsignificantly reduce (i.e., ameliorate, delay, diminish, and/ordecrease) the pathogenic effects normally seen after administration ofthe virus vaccine in the absence of IFN.

While not wishing to be held to any particular theory of the invention,it appears that effective doses of IFN protect the bird against thepathogenic effects of the virus, but allow production of an active andprotective immune response. High doses of IFN may be unsuitable in thepresent methods and pharmaceutical formulations, as they may reduce oreven block viral replication such that a protective immune response isnot induced. Thus, according to the present invention, the dose of IFNshould not be so high that a protective immune response is prevented. Itappears that there is a “window” of effective IFN doses for carrying outthe present invention. Alternatively, it appears that there is aneffective ratio of IFN to vaccine, with too low or too high an IFN dose,as compared with the dose of vaccine, being detrimental. Ranges of IFNoutside the effective window, alternatively ratios of vaccine to virusoutside of the effective range, will impede, rather than increase,vaccine efficacy.

This critical window for interferon dosage has not previously beenappreciated by the art. For example, U.S. Pat. No. 4,820,514 to Cumminsdescribes a method of vaccinating feeder cattle by oral administrationof an infectious bovine rhinotracheitis virus vaccine in conjunctionwith IFNα. However, Cummins fails to disclose that there is a window ofeffective IFN doses, or that ratios of vaccine to IFN outside of theeffective range will impede, rather than increase, vaccine efficacy.

The terms “protective immunity” or “protective immune response,” as usedherein, are intended to mean that the host bird mounts an active immuneresponse to the virus vaccine, such that upon subsequent exposure to thevirus or a virulent viral challenge, the bird is able to combat theinfection. Thus, a protective immune response will decrease theincidence of morbidity and mortality from subsequent exposure to thevirus among host birds. It is possible that with co-administration ofIFN there will be a reduction in the immune response to the virus, butthis diminishment will not be so severe that the effectiveness of thevaccine to protect the bird against future virus exposure issubstantially or totally eliminated. Those skilled in the art willunderstand that in a commercial poultry setting, the production of aprotective immune response may be assessed by evaluating the effects ofvaccination on the flock as a whole, e.g., there may still be morbidityand mortality in a minority of vaccinated birds.

By “active immune response”, it is meant any level of protection fromsubsequent exposure to the virus or virus antigens which is of somebenefit in a population of subjects, whether in the form of decreasedmortality, decreased lesions, improved feed conversion ratios, or thereduction of any other detrimental effect of the disease, and the like,regardless of whether the protection is partial or complete. An “activeimmune response” or “active immunity” is characterized by “participationof host tissues and cells after an encounter with the immunogen. Itinvolves differentiation and proliferation of immunocompetent cells inlymphoreticular tissues, which lead to synthesis of antibody or thedevelopment cell-mediated reactivity, or both.” Herbert B. Herscowitz,Immunophysiology. Cell Function and Cellular Interactions in AntibodyFormation, in Immonology: Basic Processes 117 (Joseph A. Bellanti ed.,1985). Alternatively stated, an active immune response is mounted by thehost after exposure to immunogens by infection, or as in the presentcase, by vaccination. Active immunity can be contrasted with passiveimmunity, which is acquired through the “transfer of preformedsubstances (antibody, transfer factor, thymic graft, interleukin-2) froman actively immunized host to a non-immune host.” Id.

With respect to the degree of protection provided by the interferon, thequantity of interferon administered in combination with the live virusin the vaccine need not be sufficient to provide complete protectionfrom the pathogenic effects of the virus, as long as the detrimentalresponse produced by the virus is reduced to a level at which thebenefits of the immune response produced outweigh any harm resultingfrom the vaccination. The IFN can be administered in doses as low as0.01, 0.1, 0.5, 1, 2.5, 5, 10 or 15 μg/egg, or less, and in doses ashigh as 20, 25, 30, 40, 50, 60, 70, 80, 100, 150, or even 200 μg/egg, ormore. Pharmaceutical formulations are compounded to include thesequantities of IFN per dose.

B. Virus Vaccines

The present invention is advantageously employed with live virusvaccines, preferably, vaccines containing live pathogenic viruses, i.e.,virus vaccines capable of causing disease or death in the subject if notfor the co-administration of IFN-I or IFN-II, preferably IFN-I. Thepathogenicity of the virus may be inherent in the virus itself or due tothe susceptibility of the subject to be treated (e.g., birds in ovo).Alternatively, the term “pathogenic”, as used to describe virus vaccinesherein, means that the harm caused subjects by administration of thevirus vaccine outweighs any benefit which would result therefrom. Ingeneral, more strongly pathogenic viruses (i.e., less attenuated virusesand/or non-attenuated viruses) are preferred. The virus vaccine shouldbe capable of producing an active immune response thereto in the aviansubject being treated.

As used herein, the term “live virus” refers to a virus that retains theability of infecting an appropriate subject (as opposed to inactivatedor subunit vaccines). Furthermore, as used herein, a “vaccine virus”refers to a virus that is capable of conferring protective immunity inappropriate subjects, with acceptable associated mortality andmorbidity. The term “live pathogenic virus” as used herein is intendedto exclude those live viruses (typically non-pathogenic live viruses)that have been engineered to express an antigen from a pathogenic virusor otherwise engineered to confer pathogenicity (e.g., engineered toexpress a toxin). Vaccine viruses include, e.g., commercial live virusvaccines for use in avians post-hatch. However, it must be noted thatvaccine viruses that are safe for use in post-hatch avians may beassociated with unacceptable mortality and morbidity when used in ovo.

According to the present invention, the live vaccine virus isadministered in an amount per unit dose sufficient to evoke an activeand protective immune response to the virus in the subject to betreated. It has been discovered in the course of the investigationsdescribed herein that administration of live vaccine virus inconjunction with IFN reduces the amount of virus that must be includedin the vaccine formulations to achieve a protective immune response. Aslittle as 10, 100, 1000, or even 10,000 fold lower doses of virus arerequired to induce an immune response when the virus vaccine isadministered in conjunction with IFN according to the present inventionas compared with post-hatch virus doses in the absence of IFN. The exactdose of virus to be administered in the vaccine is not critical exceptthat the dose must be effective to engender an active and protectiveimmune response by the bird. In general, depending on the inoculumadministered, the site and manner of administration, the species, ageand condition of the subject, etc., the virus dose will range from a10⁻² to 10⁷ EID₅₀ dose of virus (i.e, Embryo Infectious Dose₅₀—the doseat which 50% of vaccinated embryos are infected), more preferably a 10⁻¹to 10⁶ EID₅₀ dose of virus, yet more preferably a 10¹ to 10³ EID₅₀ doseof virus, most preferably a 10² EID₅₀ dose of virus. Pharmaceuticalformulations are compounded to include these quantities of virus perdose.

Live viruses that may be included in vaccines to be used according tothe present invention encompass any infectious avian virus, inparticular live pathogenic viruses (as defined above). Exemplaryinfectious avian viruses include, but are not limited to, rous sarcomavirus, Newcastle disease virus, infectious bursal disease virus,infectious bronchitis virus, avian infectious laryngeotracheitis virus,turkey rhinotracheitis virus, avian leukosis virus, Marek's diseasevirus, chicken anemia virus, avian influenza virus, Paramyxovirus group2-9 viruses (PMV 2-9), avipox, herpes virus of turkeys, duck enteritisvirus, Pacheco's disease virus, duck hepatitis virus, adenovirus,parvovirus, polyomavirus, pneumovirus, orthomyxovirus, coranovirus,reovirus, rotavirus, bimavirus, enterovirus, oncornavirus, arbovirus,flavovirus, and astrovirus, with Newcastle disease virus beingpreferred.

In general, in reference to the viruses specifically enumerated above,it is intended that the present invention encompass all strains of suchviruses. Viruses and strains thereof are well known in the art. See,e.g., AMERICAN ASSOCIATION OF AVIAN PATHOLOGISTS, A Laboratory Manualfor the Isolation and Identification of Pathogens (3d. ed. 1989).

The term “infectious bursal disease virus” (IBDV), as used herein,encompasses all strains of IBDV. Exemplary are the Bursal DiseaseVaccine, Lukert strain, live virus, which is obtained from eitherVineland Laboratories (Vineland, N.J.) or Salsbury Laboratories (CharlesCity, Iowa), the Bursal Disease Virulent Challenge Virus, which isobtained from the United States Department of Agriculture in Ames, Iowa(original isolate from S. A. Edgar), and Infectious Bursal Disease Virusstrain VR2161, disclosed in U.S. Pat. No. 4,824,668 to Melchior andMelson.

The term “rous sarcoma virus” (RSV), as used herein encompasses allstrains of RSV. RSV has been comprehensively studied since its discoveryearly this century. See generally 1 RNA Tumor Viruses: Molecular Biologyof Tumor Viruses 59-61 (R. Weiss et al., eds., 2d ed. 1984). Moloney (J.Nat. Cancer Inst. 16:877) reports the development of standard lots ofthe virus for use in quantitative investigations. See also, U.S. Pat.No. 3,326,767 to Holper and Kiggins. Numerous RSV strains are listed inthe American Type Culture Collection Catalogue of Animal and PlantViruses, Chlamydiae, Rickettsiae and Virus Antisera (5^(th) ed. 1986),at pages 110-112.

The term “infectious bronchitis virus” (IBV), as used herein,encompasses all strains of IBV. Exemplary strains include, but are notlimited to Mass. 41 Strain, Arkansas 99 Strain, Connecticut A5968, andMichigan State University Repository Code 42 Strain, all available fromAmerican Type Culture Collection (Rockville, Md.).

The term “adenovirus,” as used herein, encompasses all strains ofadenoviruses. Adenoviruses infect most species of turkeys and includeGroup I adenoviruses, hemorrhagic enteritis viruses, marble spleendisease viruses, the splenomegaly virus of chickens, and egg-dropsyndrome-76 (EDS-76) virus.

Finally, the term “Newcastle Disease virus”, also known as “Type IParamyxovirus” or “PMV-1”, as used herein, encompasses all strains ofNewcastle Disease virus.

C. Vaccination of Birds in ovo with Live Pathogenic Virus Vaccines

Thus, in the most preferred embodiments, the present invention providesa method of in ovo vaccination of avians by the co-administration ofIFN, preferably IFN-I, and a live pathogenic virus. The amount of IFNadministered will vary depending on the amount and type of virus beingadministered, and the developmental stage (e.g., embryonic age) andspecies of the avian being treated. However, the amount of IFN issufficient to reduce the pathogenic effects of the virus that wouldotherwise occur in the absence of IFN. The amount of IFN isinsufficient, however, to prevent the treated avian from mounting aprotective immune response. Those skilled in the art will appreciatethat other factors can be co-administered with the vaccine virus and theIFN, for example, to enhance the immune response to the virus and/or theprotective effects of the IFN.

It will also be apparent to those skilled in the art that, when treatinga plurality of avians (such as in commercial poultry production), thereduction in pathogenic effects may be assessed by evaluating theeffects of vaccination on the flock as a whole. In other words, aneffective amount of IFN used in conjunction with a pathogenic virus toimmunize a plurality of birds may still cause morbidity or mortality ina minority of birds.

D. Subjects, Modes of Administration, and Pharmaceutical Formulations

The term “avian” and “avian subjects,” as used herein, is intended toinclude males and females of any avian species, but is primarilyintended to encompass poultry which are commercially raised for eggs,meat or as pets. Accordingly, the terms “avian” and “avian subject” areparticularly intended to encompass chickens, turkeys, ducks, geese,quail, pheasant, parakeets, parrots, and the like. Chickens and turkeysare the preferred avian subjects, with chickens being most preferred.The avian subject may be a hatched bird, including newly-hatched (i.e.,about the first three days after hatch), adolescent, and adult birds.

Avian subjects may be administered interferon and vaccines of thepresent invention by any suitable means. Exemplary means are oraladministration (e.g., in the feed or drinking water), intramuscularinjection, subcutaneous injection, intravenous injection,intra-abdominal injection, eye drop, or nasal spray. Birds may also beadministered vaccines in a spray cabinet, i.e., a cabinet in which thebirds are placed and exposed to a vapor containing vaccine, or by coursespray. When administering the inventive vaccines to birds post-hatch,administration by subcutaneous injection or spray cabinet are preferred.Birds may also be administered the vaccine in ovo, as described in U.S.Pat. No. 4,458,630 to Sharma. In ovo administration of vaccine is mostpreferred. As a practical matter, it may be desirable to administercompositions including two or more vaccines to the subject at the sametime.

The in ovo administration of vaccine, as described hereinabove, involvesthe administration of the vaccine to the avian embryo while contained inthe egg. The vaccine may be administered to any suitable compartment ofthe egg (e.g., allantois, yolk sac, amnion, air cell, or into the avianembryo itself), as would apparent to one skilled in the art. Preferably,the vaccine is administered to the amnion. Eggs administered thevaccines of the present invention are fertile eggs which are preferablyin the last half, more preferably the last quarter, of incubation.Chicken eggs are treated on about the twelfth to twentieth day ofincubation, more preferably the fifteenth to nineteenth day ofincubation, and are most preferably treated on about the eighteenth dayof incubation (the eighteenth day of embryonic development). Turkey eggsare preferably treated on about the fourteenth to twenty-sixth day ofincubation, more preferably on about the twenty-first to twenty-seventhday of incubation, most preferably on about the twenty-fifth day ofincubation. Those skilled in the art will appreciate that the presentinvention can be carried out at any predetermined time in ovo, as longas the embryo is able to mount an immune response to the virus vaccine,and the IFN is able to protect the bird against the pathogenic effectsof the virus.

In preferred embodiments of the invention, chicken eggs are administereda live pathogenic Newcastle disease virus vaccine and a compositioncontaining IFN-I during the last half of in ovo incubation (preferablythe last quarter of in ovo incubation). The two administration steps maybe, but need not be, concurrent.

The IFN and vaccine can be administered concurrently, but concurrentadministration is not necessary. “Concurrently” or “concurrentadministration” is used herein to mean administration within minutes ofthe same time, not necessarily at the same precise moment. Concurrentadministration may be carried out by mixing IFN and vaccine prior toinoculation, or by simultaneous injection of the two compounds, at thesame or at different sites. Alternatively, the IFN can be injectedbefore the vaccine (even days before) to “prime” the bird prior toinoculation with the vaccine. As a further alternative, the IFN can beadministered after the vaccine has had the opportunity to infect thebird. For ease of handling in a commercial hatchery, it is preferable toadminister the IFN and virus vaccine concurrently.

If IFN is to be administered to animals concurrently with theadministration of the vaccine, the two can be administered separately ormixed together. If IFN and vaccine are mixed together prior toadministration, the vaccine formulation can be the same as standardvaccine formulations (which include a suspension of virus suitable forinducing immunity to an infectious disease), with the addition of thenecessary amount of a biologically active IFN. Such vaccine formulationsare well known to those skilled in the art. Such formulations caninclude pharmaceutically acceptable carriers, such as saline orphosphate-buffered saline (PBS).

Eggs may be administered the vaccines and IFN by any means whichtransports the compound through the shell. The preferred method ofadministration is, however, by injection. The substance may be placedwithin an extraembryonic compartment of the egg (e.g., yolk sac, amnion,allantois, air cell) or within the embryo itself. The site of injectionis preferably within the region defined by the amnion, including theamniotic fluid and the embryo itself. By the beginning of the fourthquarter of incubation, the amnion is sufficiently enlarged thatpenetration thereof is assured nearly all of the time when the injectionis made from the center of the large end of the egg along thelongitudinal axis.

The mechanism of egg injection is not critical, but it is preferred thatthe method not unduly damage the tissues and organs of the embryo or theextraembryonic membranes surrounding it so that the treatment will notdecrease hatch rate. A hypodermic syringe fitted with a needle of about18 to 22 gauge is suitable for the purpose. To inject into the air cell,the needle need only be inserted into the egg by about two millimeters.A one-inch needle, when fully inserted from the center of the large endof the egg, will penetrate the shell, the outer and inner shellmembranes enclosing the air cell, and the amnion. Depending on theprecise stage of development and position of the embryo, a needle ofthis length will terminate either in the fluid above the chick or in thechick itself. A pilot hole may be punched or drilled through the shellprior to insertion of the needle to prevent damaging or dulling of theneedle. If desired, the egg can be sealed with a substantiallybacteria-impermeable sealing material such as wax or the like to preventsubsequent entry of undesirable bacteria.

It is envisioned that a high-speed automated egg injection system foravian embryos will be particularly suitable for practicing the presentinvention. Numerous such devices are available, exemplary being thosedisclosed in U.S. Pat. Nos. 4,681,063 and 4,903,635 to Hebrank and U.S.Pat. Nos. 4,040,388, 4,469,047, and 4,593,646 to Miller. All suchdevices, as adapted for practicing the present invention, comprise aninjector containing the vaccine described herein, with the injectorpositioned to inject an egg carried by the apparatus with the vaccine.Other features of the apparatus are discussed above. In addition, ifdesired, a sealing apparatus operatively associated with the injectionapparatus may be provided for sealing the hole in the egg afterinjection thereof.

A pharmaceutical formulation of the present invention is made by mixingthe IFN, preferably IFN-I, with a vaccine in a pharmaceuticallyacceptable carrier. Pharmaceutical formulations of the present inventionpreferably comprise the vaccine and the IFN in a lyophilized form or ina pharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are preferably liquid, particularly aqueous, carriers. For thepurpose of preparing such vaccine formulations, the IFN and live virusmay be mixed in sodium phosphate-buffered saline (pH 7.4) orconventional culture media. The vaccine formulation may be stored in asterile glass container sealed with a rubber stopper through whichliquids may be injected and formulation withdrawn by syringe. Thoseskilled in the art will appreciate that pharmaceutical formulations maybe formulated containing IFN and two or more vaccine organisms. Suchmultiple vaccine formulations are advantageous because of practicalconsiderations, e.g., time, cost, minimize handling of the subject.

Vaccine formulations of the present invention may optionally contain oneor more adjuvants. Any suitable adjuvant can be used, including chemicaland polypeptide immunostimulants that enhance the immune system'sresponse to antigens. Preferably, adjuvants such as aluminum hydroxide,aluminum phosphate, plant and animal oils, and the like are administeredwith the vaccine in an amount sufficient to enhance the immune responseof the subject to the vaccine. The amount of adjuvant added to thevaccine will vary depending on the nature of the adjuvant, generallyranging from about 0.1 to about 100 times the weight of the compositioncontaining the virus, preferably from about 1 to about 10 times theweight of the composition containing the virus.

The vaccine formulations of the present invention may optionally containone or more stabilizers. Any suitable stabilizer can be used, includingcarbohydrates such as sorbitol, manitol, starch, sucrose, dextrin, orglucose; proteins such as albumin or casein; and buffers such asalkaline metal phosphate and the like.

E. Vaccine Administration to Maternal Antibody Positive Animals.

It is well-known in the veterinary, poultry and animal sciences that thepresence of maternally-transmitted antibodies in the hatchling bird oryoung mammal adversely affects vaccine efficacy. Resistance to vaccinesin young mammals and avians is a persistent problem to whichconsiderable efforts have been directed by the animal and poultryindustries. See, e.g., Kit et al., (1993) Immunology and Cell Biology71:421 (pigs); Xiang et al., (1992) Virus Res. 24:297 (mice); vanOirschot et al., (1991) J. Vet. Med. 38:391 (horses); Bjoerkholm et al.,(1995) Pediatric Infectious Disease J. 14:846 (humans); Tsukamoto etal., (1995) Avian Dis. 39:218 (chickens). The problem is particularlyacute with respect to live vaccines. Tsukamoto et al., (1995) Avian Dis.39:218. Unfortunately, there has been little success in overcoming theproblem of inactivation of vaccines by maternal antibodies. Rather, mostvaccination programs in young animals are designed to circumventmaternal antibodies by delaying vaccination until after maternalantibody levels decline or disappear.

The present investigations have led to the discovery that theadministration of IFN, in particular IFN-I, in conjunction with vaccinescan overcome the neutralizing (i.e., inhibitory or inactivating) effectsof maternal antibodies and, thus, lead to more effective vaccinationprograms for maternal antibody positive animals. Typically, the maternalantibodies neutralize, inhibit and/or inactivate the vaccine byrecognizing (i.e., specifically binding to) the vaccine immunogen. By a“maternal antibody positive” animal it is meant an animal that haspassive immunity by the transmission of maternal antibodies, i.e., fromcolostrum, milk or the egg yolk. Alternatively stated, the animals areseropositive for the vaccine organism as a result ofmaternally-transmitted antibodies. As a further alternative, a “maternalantibody positive” animal still has sufficient maternally-transmittedantibodies, such that their presence will substantially interfere withvaccine efficacy (e.g., 20%, 30%, 40%, 50%, 70%, or more), as this termis understood in the art (e.g., reduction in titers, reduction inability to withstand a challenge, and the like).

This embodiment of the invention is preferably, and advantageously,employed with vaccines that would generally be unsafe (e.g., a vaccineassociated with hatch depression). However, if lower “safe” doses ofvaccine are administered in the absence of IFN, they may not beefficacious because of the interference by maternal antibodies. Whilenot wishing to be held to any particular theory of the invention, itappears that administration of vaccine in combination with interferonaccording to the present invention, allows the administration of vaccinedoses sufficient to overcome the interfering effects of maternalantibodies. In the absence of IFN, these doses would generally result inunacceptable levels of morbidity and mortality in the host birds. TheIFN reduces the pathogenic effects of the virus, as describedhereinabove, such that higher, more efficacious, doses of vaccine can besafely administered.

Live virus vaccines are preferred, with live pathogenic virus vaccinesbeing most preferred. Vaccines and interferon for use according to thisembodiment of the invention, methods of administration thereof, andpharmaceutical formulations are as described above.

Vaccines can be administered according to the present invention to birdsin ovo and to hatchlings to administer high enough virus doses toovercome the interfering effects of maternal antibodies withoutcompromising safety. Avian subjects are as described above. In birdembryos, maternal antibodies are deposited in the yolk and are taken upby the embryo as the yolk is resorbed. Typically, maternal antibodiescan be detected in the embryo by embryonic day 15. Accordingly, thepresent invention is useful in increasing the efficacy of vaccinesadministered after embryonic day 15, more preferably after embryonic day17, to birds in ovo.

Unlike conventional vaccination methods, the inventive methods disclosedherein may be carried out to vaccinate a young bird soon after hatch. Inyoung chickens, maternal antibodies generally disappear by three weeksafter hatch. Accordingly, in young birds, vaccine and interferon areadministered within about four weeks post-hatch, preferably within aboutthree weeks post-hatch, more preferably within about two weekspost-hatch, still more preferably, within about one week post-hatch, andmost preferably within about the first three days post-hatch. Typically,vaccination will be carried out at the time that the birds aretransferred from the hatcher (usually one or two days post-hatch).

In other preferred embodiments, the invention may be practiced to moreeffectively vaccinate young mammals, even in the presence of maternalantibodies. Maternal antibodies are passed to the young mammal throughthe colostrum and, to a lesser extent milk, and disappear in the firstfew months after birth. Vaccination of young pigs by conventionalmethods, for example, is generally carried out at about three weeks ofage, about the time that maternal antibodies have disappeared and theyoung animal's own active immune responses are increasing.

The terms “mammal” and “mammalian subject”, as used herein, include themale and females of any mammalian species. Preferred are humans,domestic livestock (e.g., horses, cattle, sheep, pigs and goats, and thelike), and companion animals (e.g., cats, dogs, guinea pigs, gerbils,hamsters, and the like). Most preferred are domestic livestock species.

Any appropriate method of administering vaccines and interferon to youngmammals may be employed. Exemplary means are oral administration (e.g,by “drenching”, or by administration in the feed or drinking water),intramuscular injection, subcutaneous injection, intravenous injection,intra-abdominal injection, eye drop, or nasal spray. The young mammalmay be a neonate (i.e., about the first one to three days after birth).Alternatively, the animal may be less than about one week in age, lessthan about two weeks in age, less than about three weeks in age, lessthan about four weeks in age, less than about six weeks in age, lessthan about eight weeks in age, or less than about twelve weeks in age.Those skilled in the art will appreciate that the precise timing andmethod of administration depends on the vaccine, the age, condition andspecies of the subject, and practical and logistical considerationsrelating to the conditions in which the animal is being raised (e.g., apet dog versus a large commercial swine operation).

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof. The abbreviations usedin the Examples are defined as follows: “g” means gram, “mg” meansmilligram, “μg” means microgram, “L” means liter, “mL” means milliliter,“mol” means mole, “M” means molar, “mM” means millimolar, μM meansmicromolar, “m” means meter, “mm,” means millimeter, “nm” meansnanometer, “Da” means daltons, “kDa” means kilodaltons, “w/v” meansweight per volume, “v/v” means volume per volume, “C” means Celsius,“SPF” means specific pathogen free, “HI” means hemagglutinationinhibition, NDV means Newcastle disease virus, and “IFN” meansinterferon.

EXAMPLE 1 Materials and Methods

Recombinant chicken interferon-I (IFN-I) was expressed in the yeastPichia pastoris. Briefly, four primers (designated IFN-I through IFN-4)were designed based on the published sequence for type I IFN bySekellick et al., (1994) J. Interferon Res. 14:71. Primer IFN-3 wasdesigned as a reverse transcription primer. It is antisense to the mRNAand located 3′ to the termination of the coding region. The IFN-1 andIFN-2 primers were designed to amplify the portion of the cDNA encodingthe mature protein. Both primers contain EcoRi sites engineered onto the5′ ends to facilitate subcloning of the IFN cDNA into the Pichiapastoris pPIC9 expression vector in-frame with the secretion signalsencoded by the plasmids. Primer IFN-4 was derived from an internal cIFNmRNA sequence to facilitate sequence analysis.

Total RNA prepared from chicken splenocyte cultures was reversetranscribed with the RNA-PCR kit (Perkin-Elmer) priming with eitherrandom hexamers or primer IFN-3. PCR amplification was performed withprimers IFN-1 and IFN-2 using the RNA-PCR reagents plus 10% glycerol.Taq polymerase was added separately after preheating the other reagentsto 95° C. for 2 minutes. Amplification proceeded for 5 cycles of 95° C.,1 minute; 50° C., 2 minutes; 72° C., 1 minute; followed by 25 cycles of95° C. 1 minute; 60° C., 2 minutes; 72° C., 3 minutes. Analysis of thePCR products showed a single band of ˜500 bp. The IFN PCR product wassubcloned into the pCRII® plasmid vector (Invitrogen, Carlesbad, Calif.)according to the manufacturer's protocol. Two positive clones, selectedby restriction enzyme analysis were confirmed by DNA sequencing. Theseclones were sequenced in their entirety and were found to have no basepair changes compared with the published sequence for IFN-I.

IFN-I excised from the pCRII® vector with EcoRI was subcloned into theEcoRl site of the pPIC9 vector (Invitrogen, San Diego, Calif.) in framewith the α-F mating factor secretion signal provided in the vector.pPIC9-IFN-I, linearized by digestion with Bg/II, was isolated from softagarose and transformed into spheroplasts of the Pichia pastoris strain,GS115(HIS⁻). The yeast were plated onto minimal media for selection ofHis⁺ transformants. Transformants were then plated on selective mediathat allows identification of recombinants that have the pPIC9-IFN-IcDNA integrated into the yeast genome at the AOXI locus. Selected His⁺Mut^(S) clones were grown using standard growth and induction methods.Methanol-induced cell free supernatants of sixteen cIFN-I transformedPichia pastoris clones were media exchanged on 10 kDa centriconconcentrators (Amicon, Danvers, Mass.) and assayed in a chick embryofibroblast viral protection bioassay. Ten clones exhibited good activitycompared with controls. Bioactive IFN-I preparations were combined,concentrated and evaluated by coomassie blue and silver staining ofSDS-PAGE gels. The IFN-I banding pattern was complex with a number ofbands in the 21-45 kDa range, including a predominant band atapproximately 31 kDa. One bioactive clone was selected for scale-upproduction and evaluation of in vivo activity.

Yeast expressing the chicken IFN-I are grown using standard growth andinduction techniques. Yeast cells are removed by centrifugation, and thesupernate is clarified by microfiltration. The IFN-I is furtherprocessed by concentration and buffer exchange using a 10 kilodaltonultrafiltration membrane. In an optional step, the processed recombinantIFN-I may be further purified by reverse phase HPLC using gradientelution, and the organic mobile phase components are then removed byvacuum evaporation. The final IFN-I preparation is sterile-filtered andstored at −4° C. to −70° C., typically −20° C. to −10° C, until use.Each IFN-I batch is analyzed for protein concentration and sterility.

These studies used protein level for determining IFN-I dose.Batch-to-batch specific activity was calculated on the basis of the invitro chick embryo fibroblast viral protection bioassay. J. E. Cooliganet al., Current Protocols in Immunology, 6.9.1-6.9.3 (1995). Thespecific activity ranged from 1×10⁵ to 1×10⁸ units/mg protein. Proteindeterminations were made using the BioRad kit (Hercules, Calif.).Relative protection from IFN-I treatment was consistent among batches.

The Newcastle Disease Virus (NDV) vaccine was the B1 Type, LaSota StrainLive Virus, CLONEVAC-30 NDV vaccine from Intervet, Inc. (Millsboro,Del.). Specific Pathogen Free (SPF) leghorn eggs were obtained fromHy-Vac (Adel, Iowa). Broiler eggs (Cobb×Cobb) were obtained from CentralFarms (Fayetteville, N.C.) or from Green Forest Hatchery (Green Forest,Ark.).

Egg injection was performed on embryonic day 18 (E18) embryos byinjection into the anmion of test article in 100 μl. Confirmation ofinjection site was performed by injection of latex dye and breaking outthe embryo to visually observe site of injection. Unless notedotherwise, hatch was routinely monitored at day E22, and unhatched eggsbroken out to determine whether embryonic death was related to treatmentor not (e.g., middle death, malformed, etc.). Cumulative survivabilitywas determined at indicated time points by taking the number ofsurviving hatched chicks at a given time-point divided by the number ofeggs incubated minus death unrelated to treatments.

Statistical methods, where applicable, are indicated in the descriptionsof individual experiments.

EXAMPLE 2 Hatchability and Survivability of Chicks Vaccinated in ovowith Newcastle Disease Vaccine

This study was undertaken to investigate the relationship between in ovoNDV vaccine dose and hatchability. Treatment groups of either PBS or a10⁴, 10², 1, or 10⁻² EID₅₀ dose of NDV vaccine were administered to dayE18 embryos via amnion injection into 40 double-candled, Hyvac SPF eggsper treatment group.

Hatchability and seven-day mortality results are shown in FIG. 1 andFIG. 2, respectively. The seven-day mortality data in FIG. 2 includeembryo mortalities. Survivability results show an approximately 10%decrease in hatchability for the 1 EID₅₀ treatment group with an overall40% mortality of birds post-hatch (inclusive of embryo mortality) forthis same treatment group.

EXAMPLE 3 Assessing Safety of in ovo IFN-I Administration

An experiment was performed to determine whether IFN-I administration today E18 chick embryos is safe. Interferon-I at a dose of 0.00025, 0.025or 2.5 μg was administered to 10 eggs per treatment group andhatchability determined. As shown below on Table 1, none of the IFNtreatments resulted in hatchability less than that observed in the PBSinjected controls, indicating no overt safety problems with IFN-Iadministration at these doses.

TABLE 1 Hatchability of Chicks Administered IFN-I on Embryonic Day 18Treatment Hatchability PBS 80% 0.00025 μg IFN-I 100% 0.025 μg IFN-I 80%2.5 μg IFN-I 100%

EXAMPLE 4 Interferon -I Protects Chicks from the Lethal Effects of inovo Vaccination Against NDV

This experiment was performed to determine whether IFN-I administrationprotects chicks against the lethal effects of NDV vaccination in ovo.Day E18 eggs were administered 1 EID₅₀ dose of NDV. Birds wereco-administered PBS (vaccine control) or 0.25, 2.5 or 25 μg IFN-I. Acontrol group received PBS only, in ovo. Hatchability and survivabilityresults are shown in FIG. 3 and FIG. 4, respectively. NDV vaccinationalone resulted in approximately 30% mortality in ovo and 50% mortalitypost-hatch (8 day mortality, including lethal effects on embryo), butthese lethal effects were overcome by simultaneous administration of 25μg IFN-I (FIG. 4). The protective effects of IFN-I were dose-dependent,with more protection being observed with administration of 25 μg versus0.25 μg IFN-I.

EXAMPLE 5 Dose-Response with in ovo IFN-I: Study 1

The objective of this study was to ascertain IFN-I doses for in ovoadministration in conjunction with NDV vaccination. Day E18 eggs (25-40eggs per treatment) were injected with PBS, 10 EID₅₀ NDV, or 10 EID₅₀NDV+0.2, 2.0 or 20 μg IFN-I. Hatchability was assessed for eachtreatment group. This experimental protocol was carried out in 3separate trials. The results are shown in FIG. 5. Two outliers(treatments 2 and 4) were removed from the third trial due to technicalerror. The precision within trials is very good (CV of 2.1% and 7.7% forthe PBS and vaccine control groups, respectively).

The repeatability of the ameliorative effects of IFN-I on mortalityinduced by NDV vaccine administration was excellent in the first twotrials, but not in the third trial. Further analysis of this data,having first excluded the outliers, demonstrates a dose-dependent effectof IFN-I in ameliorating mortality from NDV vaccination in ovo. Somedegree of ameliorative effects were observed at all doses of IFN-I, with20 μg IFN-I co-administration with NDV vaccine giving the samehatchability as PBS controls.

EXAMPLE 6 Dose-Response with in ovo IFN-I: Study 2

A second IFN-I dose-response study was carried out on a single set ofbirds, essentially as described in Example 5. Day E18 eggs werevaccinated with 10 EID₅₀ dose of NDV vaccine alone, or in combinationwith 0.1, 0.2, 1.0, 2.0, 10, or 20 μg IFN-I. A non-vaccinated control(PBS), which did not receive IFN-I, was also included in theexperimental design and had 100% hatchability (data not shown). Therewere 36 eggs per treatment group. As seen in FIG. 6, IFN-I hadprotective effects on hatchability at all doses tested. A dose-dependentprotection was observed, with complete protection at 10 μg/egg andhigher.

EXAMPLE 7 Dose-Response with in ovo IFN-I: Study 3

This study evaluated higher doses of IFN-I as a follow-up to the studiespresented in Example 5 and Example 6. IFN-I at a concentration of 0, 20or 40 μg/egg was co-injected with 10 EID₅₀ NDV into day E18 eggs. Twopreparations of IFN-I were assessed. Each treatment group included 27eggs. Hatchability was determined for each treatment (FIG. 7).Protection was seen at 20 μg (both preparations) and 40 μg IFN-I. In aseparate study, yeast expressed albumin (YEA) was injected as a negativecontrol for IFN-I administration to ensure that protection is not aresult of by-products of the IFN-I expression in the yeast Pichiapastoris. No amelioration of mortality associated with NDV vaccineadministration was observed in the YEA control group (data not shown).

EXAMPLE 8 Administration of IFN-I with Increasing NDV Vaccine Dose-Study1

The purpose of this study was to determine the extent of the protectionprovided by IFN-I to SPF embryos administered larger doses of NDVvaccine. One dose (15 μg) of IFN-I was co-administered with one of threedoses of virus (10, 10², and 10³ EID₅₀ dose) to day E18 eggs (47 eggsper treatment group). Hatchability was determined for each treatmentgroup. As seen in FIG. 8, 15 μg of IFN-I was found to be protective forall virus doses. However, the degree of protection was not equivalent tothe hatchability noted in animals not receiving NDV vaccine (PBS group).In addition to the main focus of the experiment, 10 EID₅₀ dose of NDVadministered with non-HPLC purified IFN-I was compared with the same NDVdose administered with HPLC purified IFN-I for efficacy in preventingNDV vaccine-induced lethality. In this study, on a protein basis, thetwo preparations appeared equivalent in protecting embryos from NDVvaccine challenge.

EXAMPLE 9 Administration of IFN-I with Increasing NDV Vaccine Dose—Study2

This study evaluated varying doses of both NDV vaccine and IFN-I onhatchability of SPF embryos. Three doses of IFN-I (20, 30 and 50 μg/egg)were co-administered with one of three doses of virus (10, 10², and 10³EID₅₀ dose) to day E18 eggs (40 eggs/treatment). Hatchability wasdetermined for all treatment groups (FIG. 9). As seen in FIG. 9, IFN-Iwas found to be protective at 20 μg and above at all doses of NDVvaccine. Significantly, protection was extended to embryosco-administered a 10³ EID₅₀ dose of the vaccine, a dose that was 100%lethal in positive controls.

EXAMPLE 10 Survival of IFN-I Treated Chicks Vaccinated Against NDV inovo

In this study, the protective effects of IFN-I were evaluated bysurvival over 7 days post-hatch. Four doses of IFN-I (5, 15, 30 and 45μg/egg) were co-administered with a 10 EID₅₀ dose of NDV vaccine on dayE18 (60 eggs/treatment group). Assessment of hatchability indicated thatas low as 5 μg/egg of IFN-I is protective when co-administered with 10EID₅₀ dose NDV vaccine to day E18 embryos (FIG. 10). As shown in FIG.10, all doses of IFN-I showed significant protection over the entire7-day period post-hatch. Cumulative survivability, however, showed thatcomplete IFN-I protective effects (equivalent % livability tonon-“challenged” control group) lasting through the 7 day grow-outperiod were only seen in the 45 μg/embryo treatment group.

EXAMPLE 11 Cumulative Survivability Study with Increasing Vaccine Dose

Survivability data was collected for varying concentrations of IFN-I.This study was performed to examine the protective effects of IFN-I (5,10 and 20 μg/egg) on survivability when co-administered with threedifferent doses of NDV vaccine (10, 10², and 10³ EID₅₀ dose) on day E18.Each treatment group included 43 eggs. FIG. 11 illustrates datacollected from all treatment groups within the study. Some degree ofprotection was seen across all vaccine and IFN-I doses. FIG. 12 focuseson data collected over each NDV concentration dosed with 20 μg/egg ofIFN-I. Protection lasted throughout the grow-out period with 20 μg IFN-Iin animals receiving the 10² or 10 EID₅₀ doses. Animals receiving the10³ EID₅₀ dose of virus initially showed a substantial increase insurvival with 20 μg IFN-I administration (over 10³ EID₅₀ alone), butthis protective effect diminished following day 3 post-hatch.

EXAMPLE 12 Safety and Efficacy of IFN-I-NDV in SPF Embryos

A study was performed to more fully investigate the extent and theduration of both protection and protective titers in SPF animalschallenged with a virulent strain of NDV. Embryos (E18) receivedIFN-I/NDV vaccine (10 or 10² EID₅₀ dose of vaccine ±20 or 40 μg IFN-I)or relevant positive and negative controls. Each treatment group waskept under isolation conditions. The experimental design is indicated inTable 2 below. Representative groups of animals from each treatmentgroup (10 animals/treatment group) were monitored for HI titerdevelopment and weight gain. Hatchability, pre-challenge survival (%),and body weights were also determined (Table 3; % survival not inclusiveof embryonic mortality). Group 2 birds served as a control; they did notreceive the NDV vaccine in ovo, but did receive a NDV vaccine (B1,B1strain) intraocular post hatch. Surviving animals were challenged withthe Texas GB strain of NDV at 3 weeks of age (10² EID₅₀,intramuscularly). Post-challenge mortality was monitored for a period of2 weeks. Survival data shown in Table 3 indicate complete protection ofone group of animals receiving the IFN-NDV (treatment 5/5a; administeredwith 10² EID₅₀ NDV+20 μg IFN-I).

The protection data had excellent agreement with hemagglutinationinhibition titers from representative animals in each treatment groups(Table 3). From the data in Table 3, it appears that a 10² EID₅₀ dose ofNDV with 20 μg of IFN-I is safe and, most importantly, is efficacious.The IFN-I (40 μg) with 10² EID₅₀ NDV was safe for the SPF animals, butthe treatment was not efficacious (i.e., the animals were not protectedfrom a NDV challenge). It is possible that, in this instance, the 40 μgIFN-I may be so efficient at blocking viral replication that the birdsdid not develop immunity. With lower virus (10 EID₅₀ NDV) and 20 μg IFN,the two replicates were each safe, but only one replicate provedefficacious (i.e., could protect the birds against a NDV challenge). Itappears that, in some instances, one can administer too much IFN-I, sothat vaccine efficacy is impaired. However, when administered at optimalamounts of virus and IFN combinations, the vaccine is both safe andefficacious, as in treatment group 5.

TABLE 2 Experimental Treatment Groups and Hatchability Group NVaccination 1,1a 50 E18 in ovo PBS 2,2a 50 PBS at E18 PBS in ovo, NDV(B1, B1) vaccine vaccine at post hatch hatch 3,3a 250 E18 in ovo10²EID₅₀ NDV 4,4a 70 E18 in ovo 10EID₅₀NDV 5,5a 53 E18 in ovo10²EID₅₀NDV + 20 μg IFN-I 6,6a 55 E18 in ovo 10²EID₅₀NDV + 40 μg IFN-I7,7a 52 E18 in ovo 10 NDV + 20 μg IFN-I

TABLE 3 Hatch % Body Body Body Log 2 HI Log 2 Treatment % (Both SurvivalWeight Weight Weight titers HI titers % Group Groups) (day 21) (day 0)(day 21) (day 35) (Day 21) (Day 35) Protection 1 - PBS 98 100  38.1184.4 NS 0.8 NS  0 1a - PBS 100  39.2 200.4 NS 0.8 NS  0 2 - PBS + 74100  38.5 182.5 352.9 7.1 8.8 100 post hatch NDV* 2a - PBS + 100  37.7170.5 329.6 7.3 6.1 100 post hatch NDV* 3 - 10² 55 26 38.1 145.7 310.38.1 7.1  89 EID₅₀ NDV 3a - 10² 37 37.0 167.0 345.2 7.8 7.4 100 EID₅₀ NDV4 - 10 EID₅₀ 94 76 38.6 178.4 347.1 7.6 6.4 100 NDV 4a - 10 72 38.8170.4 326.1 8.0 7.1 100 EID₅₀ NDV 5 - 10² 96 88 37.5 186.3 354.6 7.3 7.4100 EID₅₀ NDV + 20 μg IFN 5a - 10² 96 37.7 178.4 361.3 7.7 8.5 100 EID₅₀NDV + 20 μg IFN 6 - 10² 86 96 38.6 191.0 NS 1.0 NS  0 EID₅₀ NDV + 40 μgIFN 6a - 10² 100  38.1 182.4 213.6 1.0 12.0   4 EID₅₀ NDV + 40 μg IFN7 - 10 EID₅₀ 89 96 37.6 192.5 NS 1.1 NS  0 NDV + 20 μg IFN 7a 10 EID₅₀100  38.1 202.8 387.3 6.1 8.6  83 NDV + 20 μg IFN *post hatch controlwas B1,B1 vaccine control.

EXAMPLE 13 Safety of IFN-I Administration in ovo to Maternal AntibodyPositive Broilers

This experiment was carried out to determine if effective NDV vaccineand IFN-I doses would be different for maternal antibody positivebroilers as compared with SPF birds. For example, maternal antibodypositive birds might require a higher virus dose and/or less IFN-I toelicit protection from the live virus vaccine. Day E18 Broiler eggs(Cobb×Cobb) were administered increasing doses of NDV vaccine (10², 10³,and 10⁴ EID₅₀ doses) in the presence and absence of 20 or 40 μg IFN-I.Note that 10² EID₅₀ dose was optimal for experiments with SPF animalsusing this batch of vaccine. As shown in FIG. 13, survival was monitoredat hatch (day 0) and during the 2-week grow-out period after hatch.

There was significant 2-week mortality when animals received any of thethree doses of NDV without IFN-I. Two-week survival was equivalent amongNDV treated birds co-administered with 20 μg IFN-I and 10² EID₅₀ dose ofvaccine and control birds receiving only PBS and vaccine in ovo. Nodifference was observed between 20 and 40 μg IFN-I co-administered witha 10³ dose of vaccine.

These results indicate that 10² EID₅₀ dose of NDV vaccine is effectivefor infecting maternal antibody positive broilers. Animals receivinghigher doses of NDV with IFN-I were protected at hatch, but theprotection did not last throughout the grow-out period. There appearedto be no benefit in giving a greater IFN-I dose for maternal antibodypositive as compared with SPF birds, i.e., 40 μg of IFN-I afforded nomore protection than did 20 μg of IFN-I.

EXAMPLE 14 Efficacy of in ovo Administration of IFN-NDV Vaccination inMaternal Antibody Positive Chickens

In order to determine whether IFN-NDV would demonstrate efficacy inmaternal antibody positive broilers when challenged with virulent NDV at4 weeks post hatch, this study inoculated embryonic day 18 broilerembryos (Cobb×Cobb) with 10² to 10³ EID₅₀ NDV in combination with 10-20μg IFN-I per egg. Controls received only PBS in ovo or 10³ EID50 NDVwithout IFN-I. There were 60 to 200 eggs per treatment group. Eachtreatment group was kept in isolation from time of injection throughgrowout. Hatchability, pre-challenge % survival, and body weights areshown (Table 4; % survival not inclusive of embryonic mortality).

As shown in Table 4, all animals tested had maternal antibodies athatch, assessed by HI titers. By 4 weeks post-hatch, maternal antibodieshad waned to non-protective levels in control animals, and protective HItiters had been established in all treatment groups receiving NDVvaccine in ovo. Although protective titers were established in the NDVtreatment group not receiving IFN-I, this vaccine dose was clearly notsafe without co-administration of IFN, as shown by the decreasedhatchability of only 87%, a significant decrease in hatchabilitycompared with the PBS controls (p≧0.05). When vaccine was administeredin the presence of IFN-I, hatchability was similar to PBS treatedcontrols.

At 4-weeks post hatch, 20 surviving birds from each treatment werechallenged with a 10² EID₅₀ NDV (Texas GB) challenge, and two-weeksurvivability was monitored. The survivability data are presented as “%Protection” in Table 4 below. All treatment groups receiving IFN-NDVcombinations in ovo were protected from virulent challenge.

The above Examples demonstrate IFN-NDV co-administration in ovo to besafe and efficacious for inducing protective immunity in SPF andmaternal antibody positive chickens.

TABLE 4 Hatch % % Body Body Body Log 2 HI Log 2 HI Log 2 HI Treatment(Both Survival Weight Weight Weight titers titers titers % Group Groups)(day 28) (day 0) (day 28) (day 42) (Day 0) (Day 28) (Day 42) Protection1 - PBS 97 100 44.0 1069.1 1759.7 3.5 0.9 9.0  5 1a - PBS 100 43.71076.4 ns 0.7 ns  0 2 - 10² EID₅₀ NDV + 97 100 43.8 1145.0 2275.1 3.56.9 7.8 100 20 μg IFN-I 2a - 10² EID₅₀ NDV +  92 44.2 1070.8 2123.1 6.67.1 100 20 μg IFN-I 3 - 10² EID₅₀ NDV + 98 100 44.5 1110.5 2213.1 3.56.4 7.5 100 10 μg IFN-I 3a - 10² EID₅₀ NDV + 100 44.0 1181.6 2340.5 6.26.8 100 10 μg IFN-I 4 - 10^(2.5) EID₅₀ NDV + 95  96 45.1 1132.5 2226.53.4 6.2 8.0 100 20 μg IFN-I 4a - 10^(2.5) EID₅₀  96 45.2 1147.7 2164.16.3 5.5 100 NDV + 20 μg IFN-I 5 - 10^(2.74) ID₅₀ NDV + 98 100 42.01118.2 2168.1 3.2 6.2 7.3 100 20 μg IFN-I 5a - 10^(2.74) EID₅₀ 100 43.01102.0 2045.6 6.2 6.3 100 NDV + 20 μg IFN-I 6 - 10³ EID 50 NDV + 97  7544.1  955.5 1975.0 3.6 6.6 6.9 100 20 μg IFN-I 6a - 10³ EID₅₀ NDV +  9244.6 1042.1 2033.5 7.5 5.9 100 20 μg IFN-I 7 - 10² EID₅₀ NDV 87  79 43.2 892.3 1963.0 3.1 7.3 6.9 100 B1 LaSota 7a - 10² EID₅₀ NDV  86 43.8 929.0 1955.8 6.6 7.0 100 B1 LaSota

EXAMPLE 15 Hatchability and Post-Challenge Survival of Maternal AntibodyPositive Commercial Broilers Vaccinated in ovo with IFN-NDV

Birds and vaccination were as described in Example 13 and Example 14.IFN-I (0, 10, 20 or 30 μg per egg) was co-administered with 0, 10^(2.5)EID₅₀ or 10^(3.5) EID₅₀ live NDV vaccine (Table 5). Hatchability oftreated embryos was monitored (FIG. 14). Birds had a mean HI titer athatch of 5.2 (Log 2) indicating a protective level of maternal antibody.Treatment groups were kept in isolation rooms until the time ofchallenge. Texas GB challenge occurred at day 28. Percent protection wasdetermined by monitoring mortality for 14 days post challenge.

The hatch data in FIG. 14 indicate that NDV-IFN-I (10^(2.5) EID₅₀+20 μgof IFN) is safe compared with in ovo NDV vaccine alone. The higher NDVvaccine dose (10³ ⁵ EID₅₀) in combination with 20 μg IFN was alsoprotective compared with in ovo NDV vaccinates alone, though not to thesame degree.

Protection from lethal challenge was shown in all of the groupsreceiving IFN-I and NDV vaccine as shown in Table 5, but not in the PBS(negative) controls. One of the PBS controls demonstrated some degree ofprotection which may have been due to resistance by the broilers in thattreatment group, or a small degree of contamination in that treatmentgroup. It should be noted that there was 100% protection in all othertreatment groups. Although protection was also observed in birds thatreceived viral vaccine without IFN-I in ovo, the viral vaccine was notsafe unless co-administered with IFN-I.

These data generated in maternal antibody positive broilers, indicatevaccination with NDV and IFN-I in ovo is safe and efficacious.

TABLE 5 Treatment replicate group # % Protected 1--PBS 41.7% 1a-PBS 8.32--10^(2.5)B1 100 LaSota, 30 μg IFN-I 2a--10^(2.5)B1 100 LaSota, 30 μgIFN-I 3--10^(2.5)B1 100 LaSota, 20 μg IFN-I 3a--10^(2.5)B1 100 LaSota,20 μg IFN-I 4--10^(2.5)B1 100 LaSota, 10 μg IFN-I 4a--10^(2.5)B1 100LaSota, 10 μg IFN-I 5--10^(3.5)B1 100 LaSota, 20 μg IFN-I 5a--10^(3.5)B1100 LaSota, 20 μg IFN-I 6--10^(2.5)B1 100 LaSota 6a--10^(2.5)B1 100LaSota 7--10^(3.5)B1 not tested LaSota 7a--10^(3.5)B1 not tested LaSota

EXAMPLE 16 Vaccination of Commercial Broilers with IFN-NDV

Safety and dose-response studies are carried out as described above todetermine the optimal (i.e., safe and efficacious) doses of both NDVvaccine and IFN-I in maternal antibody positive broilers. Commercialbroilers (Cobb×Cobb) are divided into treatment groups that receive PBSor vaccine and/or IFN-I by subcutaneous injection at various timespost-hatch (e.g., 1, 3, 7, 10 days). For ease of handling, it ispreferred to administer IFN-NDV at the time the birds are transferredfrom the hatcher, typically, one or two days after hatch. All birds arescreened for the presence of anti-NDV antibodies, by any method known inthe art, before the start of the study. Non-vaccinated control birds areisolated from vaccinated birds during the course of the study to preventinfection by virus shedding from the vaccinated birds.

Additional studies are undertaken to follow the time course of maternalantibody disappearance (i.e., antibodies against NDV) after hatch.

After optimal doses of both vaccine and IFN-I have been identified, anefficacy study is carried out as described in Example 12 and Example 14,with the exception that vaccination is post-hatch. Challenge NDV isadministered after the time when passive immunity from maternalantibodies has substantially or completely disappeared. Maternalantibody positive birds administered IFN-NDV demonstrate substantiallyimproved resistance to a virulent NDV challenge as compared with birdstreated with PBS (controls), NDV or IFN alone.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed is:
 1. A method of producing protective immunity against a viral disease in an avian subject, comprising: (a) administering to an avian subject in ovo a composition comprising a vaccine comprising a live pathogenic virus; and (b) administering to the avian subject in ovo a composition comprising interferon; wherein the live pathogenic virus is administered in an amount effective to produce an immune response in the avian subject; and wherein the interferon is administered in an amount effective to (1) reduce the pathology that would occur in the absence of the interferon due to the administration of the vaccine, and (2) allow the production of a protective immune response in the avian subject.
 2. The method according to claim 1, wherein the interferon is a Type I interferon.
 3. The method according to claim 2, wherein the Type I interferon is a chicken Type I interferon.
 4. The method according to claim 1, wherein said administering steps are carried out during the last half of in ovo incubation.
 5. The method according to claim 1, wherein said administering steps are carried out during the last quarter of in ovo incubation.
 6. The method according to claim 1, wherein said administering steps are carried out essentially concurrently.
 7. The method according to claim 6, wherein the vaccine and the interferon are included in the same composition.
 8. The method according to claim 1, wherein said administering steps are carried out by injection into the amnion of the egg.
 9. The method according to claim 1, wherein the live pathogenic virus is selected from the group consisting of rous sarcoma virus, Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus, avian infectious laryngeotracheitis virus, turkey rhinotracheitis virus, avian leukosis virus, Marek's disease virus, chicken anemia virus, avian influenza virus, Paramyxovirus group 2-9 viruses (PMV 2-9), avipox, herpes virus of turkeys, duck enteritis virus, Pacheco's disease, duck hepatitis virus, adenovirus, parvovirus, polyomavirus, pneumovirus, orthomyxovirus, coranovirus, reovirus, rotavirus, birnavirus, enterovirus, oncornavirus, arbovirus, flavovirus, and astrovirus.
 10. The method according to claim 1, wherein the live pathogenic virus is a Newcastle disease virus.
 11. The method according to claim 1, wherein the avian subject is administered about a 10⁻² EID₅₀ to about a 10⁶ EID₅₀ dose of the live pathogenic virus.
 12. The method according to claim 1, wherein the avian subject is selected from the group consisting of chickens, turkeys, ducks, geese, quail and pheasant.
 13. The method according to claim 1, wherein the avian subject is a chicken.
 14. The method according to claim 1, wherein the avian subject has maternal antibodies that recognize the live pathogenic virus.
 15. A method of producing protective immunity against Newcastle disease in a chicken, comprising: (a) administering to a chicken during the last half of in ovo incubation a composition comprising a vaccine comprising a live pathogenic Newcastle disease virus; and (b) administering to a chicken during the last half of in ovo incubation a composition comprising a Type I interferon; wherein the live pathogenic virus is administered in an amount effective to produce an immune response in the chicken; and wherein the Type I interferon is administered in an amount effective to (1) reduce the pathology that would occur in the absence of the Type I interferon due to the administration of the vaccine, and (2) allow the production of a protective immune response in the chicken.
 16. A method of reducing mortality from the administration of a live vaccine virus in ovo to an avian subject, comprising: (a) administering to an avian subject in ovo a composition comprising a vaccine comprising a live vaccine virus; and (b) administering to the avian subject in ovo a composition comprising interferon; wherein the live vaccine virus is administered in an amount effective to produce an immune response in the avian subject; and wherein the interferon is administered in an amount effective to (1) reduce the pathology that would occur in the absence of the interferon due to the administration of the vaccine, and (2) allow the production of a protective immune response in the avian subject.
 17. The method according to claim 16, wherein the interferon is a Type I interferon.
 18. The method according to claim 17, wherein the Type I interferon is a chicken Type I interferon.
 19. The method according to claim 16, wherein said administering steps are carried out during the last quarter of in ovo incubation.
 20. The method according to claim 16, wherein the vaccine and the Type I interferon are included in the same composition.
 21. The method according to claim 16, wherein the live pathogenic virus is selected from the group consisting of rous sarcoma virus, Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus, avian infectious laryngeotracheitis virus, turkey rhinotracheitis virus, avian leukosis virus, Marek's disease virus, chicken anemia virus, avian influenza virus, Paramyxovirus group 2-9 viruses (PMV 2-9), avipox, herpes virus of turkeys, duck enteritis virus, Pacheco's disease, duck hepatitis virus, adenovirus, parvovirus, polyomavirus, pneumovirus, orthomyxovirus, coranovirus, reovirus, rotavirus, birnavirus, enterovirus, oncornavirus, arbovirus, flavovirus, and astrovirus.
 22. The method according to claim 16, wherein the live pathogenic virus is a Newcastle disease virus.
 23. The method according to claim 16, wherein the avian subject is administered about a 10⁻² EID₅₀ to about a 10⁶ EID₅₀ dose of the live pathogenic virus.
 24. The method according to claim 16, wherein the avian subject is selected from the group consisting of chickens, turkeys, ducks, geese, quail and pheasant.
 25. The method according to claim 16, wherein the avian subject is a chicken.
 26. The method according to claim 16, wherein the avian subject has maternal antibodies that recognize the live pathogenic virus.
 27. A method of producing protective immunity against a viral disease in an avian subject, comprising: (a) administering to an avian subject in ovo a composition comprising a vaccine comprising a live pathogenic virus; and (b) administering to the avian subject in ovo a composition comprising about 1 μg to about 80 μg of a Type I interferon, said Type I interferon having a specific activity of 1×10⁵ to 1×10⁸ units per milligram; wherein the live pathogenic virus is administered in an amount effective to produce an immune response in the avian subject; and wherein the interferon is administered in an amount effective to (1) reduce the pathology that would occur in the absence of the interferon due to the administration of the vaccine, and (2) allow the production of a protective immune response in the avian subject.
 28. The method according to claim 27, wherein the avian subject is administered about 10 μg to about 40 μg of the Type I interferon.
 29. A method of reducing mortality from the administration of a live vaccine virus in ovo to an avian subject, comprising: (a) administering to an avian subject in ovo a composition comprising a vaccine comprising a live vaccine virus; and (b) administering to the avian subject in ovo a composition comprising about 10 μg to about 40 μg of a Type I interferon, said Type I interferon having a specific activity of 1×10⁵ to 1×10⁸ units per milligram; wherein the live vaccine virus is administered in an amount effective to produce an immune response in the avian subject and an amount effective to produce mortality in the absence of the interferon; and wherein the interferon is administered in an amount effective to (1) reduce the pathology that would occur in the absence of the interferon due to the administration of the vaccine, and (2) allow the production of a protective immune response in the avian subject.
 30. The method according to claim 27, wherein the Type I interferon is a chicken Type I interferon.
 31. The method according to claim 27, wherein said administering steps are carried out during the last half of in ovo incubation.
 32. The method according to claim 27, wherein said administering steps are carried out during the last quarter of in ovo incubation.
 33. The method according to claim 27, wherein said administering steps are carried out essentially concurrently.
 34. The method according to claim 33, wherein the vaccine and the interferon are included in the same composition.
 35. The method according to claim 27, wherein said administering steps are carried out by injection into the amnion of the egg.
 36. The method according to claim 27, wherein the live pathogenic virus is selected from the group consisting of rous sarcoma virus, Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus, avian infectious laryngeotracheitis virus, turkey rhinotracheitis virus, avian leukosis virus, Marek's disease virus, chicken anemia virus, avian influenza virus, Paramyxovirus group 2-9 viruses (PMV 2-9), avipox, herpes virus of turkeys, duck enteritis virus, Pacheco's disease, duck hepatitis virus, adenovirus, parvovirus, polyomavirus, pneumovirus, orthomyxovirus, coranovirus, reovirus, rotavirus, birnavirus, enterovirus, oncornavirus, arbovirus, flavovirus, and astrovirus.
 37. The method according to claim 27, wherein the live pathogenic virus is a Newcastle disease virus.
 38. The method according to claim 27, wherein the avian subject is administered about a 10⁻² EID₅₀ to about a 10⁶ EID₅₀ dose of the live pathogenic virus.
 39. The method according to claim 27, wherein the avian subject is selected from the group consisting of chickens, turkeys, ducks, geese, quail and pheasant.
 40. The method according to claim 27, wherein the avian subject is a chicken.
 41. The method according to claim 27, wherein the avian subject has maternal antibodies that recognize the live pathogenic virus.
 42. A method of producing protective immunity against Newcastle disease in a chicken, comprising: (a) administering to a chicken during the last half of in ovo incubation a composition comprising a vaccine comprising about a 10⁻² EID₅₀to about a 10⁶ EID₅₀ dose of a live pathogenic Newcastle disease virus; and (b) administering to a chicken during the last half of in ovo incubation a composition about 10 μg to about 40 μg of a Type I interferon, said Type I interferon having a specific activity of 1×10⁵ to 1×10⁸ units per milligram; wherein the live pathogenic virus is administered in an amount effective to produce an immune response in the chicken; and wherein the Type I interferon is administered in an amount effective to (1) reduce the pathology that would occur in the absence of the Type I interferon due to the administration of the vaccine, and (2) allow the production of a protective immune response in the chicken.
 43. A method of producing protective immunity against a pathogenic viral disease in an avian subject, comprising: (a) administering to an avian subject in ovo a composition comprising a vaccine comprising a live pathogenic virus; and (b) administering to the avian subject in ovo a composition comprising interferon; wherein the live pathogenic virus is administered in an amount effective to produce an immune response in the avian subject and in an amount sufficient to cause at least about a 30% decrease in hatch rate in specific pathogen free avian subjects in the absence of the interferon; and wherein the interferon is administered in an amount effective to (1) reduce the pathology that would occur in the absence of the interferon due to the administration of the vaccine, and (2) allow the production of a protective immune response in the avian subject. 